Analytical instrument and method for evaluating microbial contamination of an object

ABSTRACT

An instrument and method for ascertaining a viable aerobic microbe count at t o  employing a two-point calibration curve of t threshold  to TVC for each type of sample. One point on the calibration curve is the x-intercept value (i.e., an estimated or experimental value for the logarithm of the minimum viable aerobic microbe count at commencement of testing (t o ) in a sample effective for causing the sample to reach t Threshold  substantially instantaneously upon commencement of incubation). The other point is ascertained experimentally from a sample having a smaller known viable aerobic microbe count at t o .

This application claims the benefit of U.S. Provisional Application No.61/477,695, filed Apr. 21, 2011.

BACKGROUND

The Total Viable Count (TVC) of aerobic bacteria in a sample can bedetermined by monitoring depletion of oxygen in the sample over timeuntil a threshold value is reached based upon a previously establishedcalibration curve of tthreshold to TVC. See, for example, F. C. O'Mahonyand D. B. Papkovsky, Rapid High-Throughput Assessment of AerobicBacteria in Complex Samples by Fluorescence-Based Oxygen Respirometry,Applied and Environmental Microbiology, vol. 72, No. 2, February 2006,pp. 1279-1287.

The calibration curves of t_(threshold) to TVC are established byexperimentally determining t_(threshold) for several samples containingknown different starting TVCs, plotting the results in semilogarithmicscale and establishing a linear equation for the regression line.

While such systems are relatively quick, easy and accurate, the time andexpense involved in establishing a calibration curve of t_(threshold) toTVC for each type of sample (e.g., each SKU or each type of swab orswab-sampling protocol) renders the system unsuitable for commercialuse, particularly for companies with an ever changing product lineand/or with dozens or even thousands of different SKUs.

Accordingly, a substantial need exists for a system and method ofquickly and inexpensively obtaining an accurate calibration curve oft_(threshold) to TVC for each type of sample.

SUMMARY OF THE INVENTION

I have surprisingly discovered that an accurate calibration curve oft_(threshold) to TVC for each type of sample can be established fromjust two-points, wherein one point is the x-intercept value (i.e., anestimated or experimental value for the logarithm of the minimum viableaerobic microbe count at commencement of testing (t_(o)) in a sampleeffective for causing the sample to reach t_(Threshold) substantiallyinstantaneously upon commencement of incubation), and the other point isascertained experimentally from a sample having a smaller known viableaerobic microbe count at t_(o), preferably a viable aerobic microbecount at t_(o) which is several orders of magnitude smaller than t_(o)at the x-intercept.

A first aspect of the invention is a method for evaluating microbialcontamination of an object, comprising the steps of: (a) incubating asample taken from the object starting at time t_(o), (b) periodicallyascertaining an O₂ concentration of the incubating sample until thedetected O₂ concentration decreases below a threshold value, (c)establishing the time period t_(Threshold) measured from t_(o) to thetime at which the O₂ concentration decreased below the threshold value,and (d) correlating t_(Threshold) to a viable aerobic microbe count inthe sample prior to incubation, utilizing the linear equation:y=mx+b  (Eq. 1)

-   -   wherein:    -   x=logarithm of the viable aerobic microbe count per unit weight        of, volume of or area swabbed to obtain a sample at t_(o)    -   y=t_(Threshold)    -   x-intercept=(−b/m)=an estimated or experimental value for the        logarithm of the minimum viable aerobic microbe count at t_(o)        in a sample effective for causing the sample to reach        t_(Threshold) substantially instantaneously upon commencement of        incubation, and        m(slope)=(y ₂ −y ₁)/(x ₂ −x ₁) wherein:        -   x₁ and y₁ are ascertained experimentally by establishing            t_(Threshold) for a sample taken from an object having a            known x, and        -   x₂ and y₂ are values of x and y at the x-intercept (−b/m,            0).

The sample is preferably incubated in a hermetically sealed chamber inthe presence of an oxygen sensitive photoluminescent dye with O₂concentration of the incubating sample ascertained by measuringfluorescence of the oxygen sensitive dye.

A second aspect of the invention is an analytical instrument comprisingan O₂ sensor and a microprocessor in electrical communication with thesensor, wherein the instrument is effective for (i) periodicallyascertaining an O₂ concentration of an incubating sample taken from anobject, (ii) determining a time period t_(Threshold) from a time t_(o)at which incubation of the sample commenced to a time at which anascertained O₂ concentration for the sample first falls below athreshold value, (iii) correlating t_(Threshold) to a viable aerobicmicrobe count in the sample prior to incubation, utilizing the linearequation:y=mx+b  (Eq. 1)

-   -   wherein:    -   x=logarithm of the viable aerobic microbe count per unit weight        of, volume of or area swabbed to obtain a sample at t_(o)    -   y=t_(Threshold)    -   x-intercept=(−b/m)=an estimated or experimental value for the        logarithm of the minimum viable aerobic microbe count at t_(o)        in a sample effective for causing the sample to reach        t_(Threshold) substantially instantaneously upon commencement of        incubation, and        m(slope)=(y ₂ −y ₁)/(x ₂ −x ₁) wherein:        -   x₁ and y₁ are ascertained experimentally by establishing            t_(Threshold) for a sample taken from an object having a            known x, and        -   x₂ and y₂ are values of x and y at the x-intercept (−b/m, 0)            and    -   (iv) transmitting a correlated value of x to a peripheral        device.

The O₂ sensor preferably comprises an oxygen sensitive photoluminescentdye having a luminescent lifetime and a reader effective for determiningO₂ concentration in an environment in fluid communication with the dyeby measuring fluorescence of the oxygen sensitive dye.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exemplary calibration curve prepared in accordance with thepresent invention.

FIG. 2 is an exemplary O₂ sensor and microprocessor taking a readingfrom an incubating sample in accordance with the present inventionwherein the sample is a liquefied specimen detached from the objectbeing evaluated.

FIG. 3 is an exemplary incubating sample in accordance with the presentinvention wherein the sample is a swab-sampling taken from the surfaceof an object being evaluated.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT Nomenclature

-   10 Interrogation Device-   15 Display Component of Interrogation Device-   16 Microprocessor-   20 Vessel, Vial or Cuvette-   21 Open Top End of Vessel, Vial or Cuvette-   22 Closed Bottom End of Vessel, Vial or Cuvette-   29 Retention Chamber of Vessel, Vial or Cuvette-   30 Probe-   40 Growth Media-   50 Sample-   51 Swab containing Sample    Construction

The invention is directed to an instrument and method for evaluatingmicrobial contamination of an object (not shown) by monitoring changesin oxygen concentration in an incubating sample 50 taken from the object(not shown) and correlating observed changes to a viable aerobic microbecount in the sample 50 prior to incubation employing a readilycalibratable linear equation.

The object (not shown) to be evaluated for microbial contamination isessentially unlimited, ranging from solids such as equipment used inmeat processing and packaging facilities, food preparation countertops,medical instruments, fruits, vegetables and meats, and liquids such asdairy products, water retained within a water tower, biological samples,sewage treated by a sewage treatment system, water flowing in rivers andstreams, etc.

Persons of routine skill in the art are capable of obtaining andpreparing suitable samples 50 from the various objects (not shown) ofinterest, including specifically but not exclusively detaching aspecimen from a solid object (not shown) such as a chicken breast, aswab-sampling taken from a hard surface such as a sanitized stainlesssteel countertop used in a food preparation area, an aliquot taken fromsewage treatment facility, etc.

Each sample 50 is placed within the retention chamber 29 of a vessel 20having an open top end 21, a closed bottom end 22, and a probe 30 withinthe retention chamber 29. The vessel 20 is preferably a vial or cuvettehaving a high aspect ratio of depth to circumference, such as disclosedin United States Patent Application Publication 2009/0029402, whichdisclosure is incorporated herein by reference. Since a preferred vessel20 is a vial or cuvette 20, the balance of the disclosure shallreference the vessel 20 as a vial 20 without intending to be limitedthereby.

The preferred methods and instruments described herein monitor microbialconsumption of oxygen (O₂) in a sample 50 based upon the quenching ofphotoluminescence by oxygen (O₂). Luminescence encompasses bothfluorescence and phosphorescence. Electromagnetic radiation in theultraviolet or visible region is used to excite molecules to higherelectronic energy levels. The excited molecules lose their excess energyby one of several methods. One of those methods is fluorescence.Fluorescence refers to the radiative transition of electrons from thefirst excited singlet state to the singlet ground state (S₁. to S₀). Thelifetime of fluorescence is relatively short, approximately 10⁻⁹ to 10⁻⁷seconds. However, intersystem crossing from the lowest excited singletstate to the triplet state often occurs and is attributed to thecrossing of the potential energy curves of the two states. The tripletstate so produced may return to the ground state by a radiative processknown as phosphorescence. Phosphorescence is the radiative relaxation ofan electron from the lowest excited triplet state to the singlet groundstate (T₁ to S₀). Because the transition that leads to phosphorescenceinvolves a change in spin multiplicity, it has a low probability andhence a relatively long lifetime of 10⁻⁴ to 10 seconds. Fluorescent andphosphorescent lifetime is known to change in a defined fashion relativeto changes in the partial pressure of an analyte (P_(A)) capable ofquenching the photoluminescent molecules. Hence, the P_(A) in fluidcommunication with a photoluminescent material can be determined bymeasuring photoluminescence lifetime.

In a preferred embodiment, the probes 30 are optically-active, oxygenpartial pressure sensitive materials configured and arranged toexperience changes in oxygen partial pressure P_(O2) in a sample 50placed within the retention chamber 29 of a vial 20. Theoxygen-sensitive material is preferably a photoluminescent dye embeddedwithin an oxygen permeable polymer matrix. Alternatively, theoxygen-sensitive photoluminescent dye can be dissolved in the sample 50and/or any added growth media 40.

The oxygen-sensitive photoluminescent dye may be selected from any ofthe well-known oxygen sensitive photoluminescent dyes. One of routineskill in the art is capable of selecting a suitable dye based upon theintended use of the probe. A nonexhaustive list of suitable oxygensensitive photoluminescent dyes includes specifically, but notexclusively, ruthenium(II)-bipyridyl andruthenium(II)-diphenylphenanothroline complexes, porphyrin-ketones suchas platinum(II)-octaethylporphine-ketone, platinum(II)-porphyrin such asplatinum(II)-tetrakis(pentafluorophenyl)porphine,palladium(II)-porphyrin such aspalladium(II)-tetrakis(pentafluorophenyl)porphine, phosphorescentmetallocomplexes of tetrabenzoporphyrins, chlorins, azaporphyrins, andlong-decay luminescent complexes of iridium(III) or osmium(II).

Typically, the hydrophobic oxygen-sensitive photoluminescent dye iscompounded with a suitable oxygen-permeable and hydrophobic carriermatrix. Again, one of routine skill in the art is capable of selecting asuitable oxygen-permeable hydrophobic carrier matrix based upon theintended use of the probe 30 and the selected dye. A nonexhaustive listof suitable polymers for use as the oxygen-permeable hydrophobic carriermatrix includes specifically, but not exclusively, polystryrene,polycarbonate, polysulfone, polyvinyl chloride and some co-polymers.

When the probe 30 is based on the quenching of photoluminescence by ananalyte, the vial 20, or at least that portion of the vial 20 coatedwith the probe 30, must allow radiation at the excitation and emissionwavelengths to be transmitted to and received from the probe 30 withminimal interference.

Instruments 10 for interrogating probes 30 based on the quenching ofphotoluminescence by an analyte are well known and commerciallyavailable from various sources, including Becton Dickinson of FranklinLakes, N.J. and Mocon, Inc. of Minneapolis, Minn. Such instruments 10typically include or are equipped to communicate with a microprocessor16, computer memory (not shown), an input device such as a mouse (notshown) and/or a keyboard (not shown), a display 15 and a printer (notshown).

Manufacture of Probe 30 Containing Vials 20

The probe 30 containing vials 20 can be conveniently manufactured by (A)preparing a coating cocktail (not shown) which contains thephotoluminescent oxygen-sensitive dye and the oxygen-permeable polymerin an organic solvent (not shown) such as ethylacetate, (B) depositingthe cocktail into the bottom 22 of the retention chamber 29, such as byusing a syringe (not shown), and (C) allowing the cocktail (not shown)to dry, whereby a solid-state coating is formed within the retentionchamber 29 at the bottom 22 of the vial 20, thereby forming a probe 30within the vial 20.

Generally, the concentration of the polymer in the organic solventshould be in the range of 0.1 to 20% w/w, with the ratio of dye topolymer in the range of 1:20 to 1:10,000 w/w, preferably 1:50 to 1:5,000w/w.

Use

Microbial contamination of an object can be evaluated by (a) obtainingand depositing a sample 50 taken from the object into the retentionchamber 29 of a vessel 20 equipped with a probe 30, (b) optionallydigesting or stomaching the sample 50 for a defined period of time, (c)optionally depositing growth media 40 into the retention chamber 29 whennecessary and appropriate for supporting growth of microbes containedwithin the deposited sample 50, (d) incubating the sample 50 starting attime t_(o), (e) periodically ascertaining an O₂ concentration of theincubating sample 50 until the detected O₂ concentration at the probe 30decreases below a threshold value, (f) establishing the time periodt_(Threshold) measured from t_(o) to the time at which the O₂concentration decreased below the threshold value, and (g) correlatingt_(Threshold) to a viable aerobic microbe count in the sample prior toincubation, utilizing the linear equation:y=mx+b  (Eq. 1)

-   -   wherein:    -   x=logarithm of the viable aerobic microbe count per unit weight        of, volume of or area swabbed to obtain a sample at t_(o)    -   y=t_(Threshold)    -   x-intercept=(−b/m)=an estimated or experimental value for the        logarithm of the minimum viable aerobic microbe count at t_(o)        in a sample effective for causing the sample to reach        t_(Threshold) substantially instantaneously upon commencement of        incubation, and        m(slope)=(y ₂ −y ₁)/(x ₂ −x ₁) wherein:        -   x₁ and y₁ are ascertained experimentally by establishing            t_(Threshold) for a sample taken from an object having a            known x, and        -   x₂ and y₂ are values of x and y at the x-intercept (−b/m,            0).

The sample is preferably incubated in a hermetically sealed chamber inthe presence of an oxygen sensitive photoluminescent dye with O₂concentration of the incubating sample ascertained by measuringfluorescence of the oxygen sensitive dye with an analytical instrument10 comprising an O₂ sensor and a microprocessor in electricalcommunication with the sensor.

The O₂ concentration threshold value may be established as (i) a %decrease in the atmospheric concentration of O₂, (ii) a % decrease inthe concentration of O₂ in fluid communication with the probe 30 in thevessel 20 relative to the O₂ concentration in fluid communication withthe probe 30 in the vessel 20 at t_(o), or (iii) an absolute value ofthe concentration of O₂ within the retention chamber 29 of the vessel20. When either option (i) or option (ii) is employed a preferred %decrease is a decrease of between 25% and 75%. When option (iii) isemployed an absolute value of between 5% and 16% is preferred.

When the sample 50 is digested or stomached, t_(o) should be establishedafter the sample 50 has been stomached for a defined period of timet_(Stomach). This defined period of time is preferably set to occurwithin the lag phase growth period of bacteria within the sample 50,with a time period of less than 60 minutes typically acceptable, and atime period of less than 5 minutes often desired.

The oxygen concentration within the retention chamber 29 can be checkedon substantially any desired scheduled with readings spaced more thantwo hours apart tending to produce results subject to excessiveinaccuracy in the final ascertained value for TVC at t_(o) whilereadings spaced less than ½ hour apart require substantial additionaltime and effort with only modest improvement in accuracy of the finalascertained value for TVC at t_(o).

The radiation emitted by the excited probe 30 can be measured in termsof intensity and/or lifetime (rate of decay, phase shift or anisotropy),with measurement of lifetime generally preferred as a more accurate andreliable measurement technique when seeking to establish oxygenconcentration via measurement of the extent to which the dye has beenquenched by oxygen.

The Linear Equation and Calibration Thereof

In order to determine Total Viable Count (TVC) of aerobic bacteria in asample 50 from the time required for the hermetically sealed sample 50to consume a defined quantity or % of oxygen requires use of apreviously established calibration curve of t_(threshold) to TVC such asdepicted in FIG. 1.

Traditionally, such calibration curves of t_(threshold) to TVC areestablished by experimentally determining t_(threshold) for severalsamples 50 of the same sample type (e.g., ground water, milk, chickenbreast, ground hamburger, ground egg shells, swab sample taken from theexterior of an unbroken egg, swab sample taken from a sanitizedstainless steel food processing work surface, etc.) containing knowndifferent starting TVCs, plotting the results in semilogarithmic scaleand establishing a linear equation for the regression line.

I have surprisingly discovered that an accurate calibration curve of TVCfor t_(threshold) to each type of sample can be established from justtwo-points, wherein one point is the x-intercept value (i.e., anestimated or experimental value for the logarithm of the minimum viableaerobic microbe count at commencement of testing (t_(o)) in a sample 50effective for causing the sample 50 to reach t_(Threshold) substantiallyinstantaneously upon commencement of incubation), and the other point isascertained experimentally from a sample 50 having a smaller knownviable aerobic microbe count at t_(o), preferably a viable aerobicmicrobe count at t_(o) which is several orders of magnitude smaller thant_(o) at the x-intercept. The logarithms employed are preferably naturallogarithms.

Once a calibration curve of t_(threshold) to TVC has been establishedfor a sample type, a viable aerobic microbe count in a sample 50 of thatsample type prior to incubation can be ascertained from anexperimentally obtained value for t_(Threshold) measured from t_(o) tothe time at which the O₂ concentration decreased below a threshold valueutilizing the linear equation:y=mx+b  (Eq. 1)

-   -   wherein:    -   x=logarithm of the viable aerobic microbe count per unit weight        of, volume of or area swabbed to obtain a sample at t_(o)    -   y=t_(Threshold)    -   x-intercept=(−b/m)=an estimated or experimental value for the        logarithm of the minimum viable aerobic microbe count at t_(o)        in a sample effective for causing the sample to reach        t_(Threshold) substantially instantaneously upon commencement of        incubation, and        m(slope)=(y ₂ −y ₁)/(x ₂ −x ₁) wherein:        -   x₁ and y₁ are ascertained experimentally by establishing            t_(Threshold) for a sample taken from an object having a            known x, and        -   x₂ and y₂ are values of x and y at the x-intercept (−b/m,            0).

The x-intercept value is preferably established experimentally, but canbe estimated. When estimated, an estimated value of between 6 and 10,most often between 7 and 9, has been found to produce acceptable resultsin most situations.

EXAMPLES Example 1 Prophetic

Detached Sample

An interrogation device 10 capable of determining O₂ concentration in asample 50 in fluid communication with a probe 30 by measuringfluorescence of the oxygen sensitive dye in the probe 30 is providedwith values for m (slope) and b (y-intercept) for various specifiedtypes of samples, whereby Equation 1 can be solved for x (logarithm ofthe viable aerobic microbe count per unit weight of, volume of or areaswabbed to obtain a sample at t_(o)) once y (t_(Threshold)) isexperimentally obtained from a sample 50 of one of the specified typesof samples 50.

Samples 50 of a processed food product containing a preservative,matching a type of sample 50 for which the interrogation device 10 has avalue for m and b, are taken from a processing line every hour on thehalf hour to ascertain viable bacterial count (TVC, APC or CFU) justprior to packaging. Each sample 50 is transported from the processingline to a testing room, digested for a predefined period of time, and aknown volume or weight of the sample 50 deposited into a barcoded vial20 containing a photoluminescent oxygen-sensitive probe 30.

An initial reading is taken from the probe 30 in each vial 20 containinga sample 50 by interrogating the probe 30 with an interrogation device10. The type of sample 50, results of the initial reading, and the timeof day at which the initial reading is taken are recorded and correlatedto the barcode of the tested vial 20. Upon completion of the initialinterrogation a schedule is established for subsequent interrogations ofeach vial 20 and the vials 20 are placed into an incubation chamber (notshown).

Each vial 20 is subsequently interrogated on the establishedinterrogation schedule. The results of each interrogation and elapsedtime since the initial interrogation for each vial 20 are recorded.

Each vial 20 is interrogated until a threshold value is reached in thereadings from the probe 30, at which time the interrogation device 10indicates that testing is complete, determines viable bacterial count(x) employing the previously provided values for m and b for the type ofsample 50 tested and the experimentally ascertained value for y, andprovides the operator with an indication of whether the sample 50contained an ACCEPTABLE or UNACCEPTABLE viable bacterial count basedupon a preestablished threshold value for x. Upon request, the operatorcan obtain the actual value of the viable bacterial count from theinterrogation device 10.

Example 2 Prophetic

Swabbed Sample

An interrogation device 10 capable of determining O₂ concentration in asample 50 in fluid communication with a probe 30 by measuringfluorescence of the oxygen sensitive dye in the probe 30 is providedwith values for m (slope) and b (y-intercept) for various specifiedtypes of samples, whereby Equation 1 can be solved for x (logarithm ofthe viable aerobic microbe count per unit weight of, volume of or areaswabbed to obtain a sample at t_(o)) once y (t_(Threshold)) isexperimentally obtained from a sample 50 of one of the specified typesof samples 50.

Samples 50, obtained by swabbing known areas of a work surface in a foodprocessing plant, matching a type of sample 50 for which theinterrogation device 10 has a value for m and b, are taken each time thework surface is cleaned and sanitized to ascertain viable bacterialcount (TVC, APC or CFU) and thereby verify the effectiveness of thecleaning and sanitization process. The sample-containing portion of theswabs 51 are immediately deposited into a barcoded vial 20 containing aphotoluminescent oxygen-sensitive probe 30 and a suitable growth medium40 added.

An initial reading is taken from the probe 30 in each vial 20 containinga sample 50 by interrogating the probe 30 with an interrogation device10. The type of sample 50, results of the initial reading, and the timeof day at which the initial reading is taken are recorded and correlatedto the barcode of the tested vial 20. Upon completion of the initialinterrogation a schedule is established for subsequent interrogations ofeach vial 20 and the vials 20 are placed into an incubation chamber (notshown).

Each vial 20 is subsequently interrogated on the establishedinterrogation schedule. The results of each interrogation and elapsedtime since the initial interrogation for each vial 20 are recorded.

Each vial 20 is interrogated until a threshold value is reached in thereadings from the probe 30, at which time the interrogation device 10indicates that testing is complete, determines viable bacterial count(x) employing the previously provided values for m and b for the type ofsample 50 tested and the experimentally ascertained value for y, andprovides the operator with an indication of whether the sample 50contained an ACCEPTABLE or UNACCEPTABLE viable bacterial count basedupon a preestablished threshold value for x. Upon request, the operatorcan obtain the actual value of the viable bacterial count from theinterrogation device 10.

We claim:
 1. A method for evaluating microbial contamination of anobject, comprising the steps of: (a) incubating a sample taken from theobject starting at time t_(o), (b) periodically ascertaining an O₂concentration of the incubating sample until the detected O₂concentration decreases below a threshold value, (c) establishing thetime period t_(Threshold) measured from t_(o) to the time at which theO₂ concentration decreased below the threshold value, and (d)correlating t_(Threshold) to a viable aerobic microbe count in thesample prior to incubation, utilizing the linear equation:y=mx+b  (Eq. 1) wherein: x=logarithm of the viable aerobic microbe countper unit weight of, volume of or area swabbed to obtain a sample att_(o) y=t_(Threshold) x-intercept=(−b/m)=an estimated or experimentalvalue for the logarithm of the minimum viable aerobic microbe count att_(o) in a sample effective for causing the sample to reacht_(Threshold) substantially instantaneously upon commencement ofincubation, andm(slope)=(y ₂ −y _(l))/(x ₂ −x ₁) wherein: x₁ and y_(l) are ascertainedexperimentally by establishing t_(Threshold) for a sample taken from anobject having a known x, and x₂ and y₂ are values of x and y at thex-intercept (−b/m, 0).
 2. The method of claim 1 wherein the logarithmsare natural logarithms.
 3. The method of claim 1 wherein the sample is aspecimen detached from the object and x=logarithm of the viable aerobicmicrobe count per unit weight or volume of a sample at t_(o).
 4. Themethod of claim 1 wherein the sample is a swab-sampling taken from thesurface of the object and x=logarithm of the viable aerobic microbecount per unit area of the object swabbed to obtain a sample at t_(o).5. The method of claim 4 wherein the swab-sampling is taken after thesurface has been sanitized for purposes of validating sanitization ofthe surface.
 6. The method of claim 3 wherein the object is a foodstuffand t_(o) occurs after the sample has been stomached for a definedperiod of time t_(Stomach).
 7. The method of claim 6 wherein t_(Stomach)is completed within a lag phase growth of the bacteria.
 8. The method ofclaim 1 wherein an O₂ concentration of the incubating sample isascertained at least once every hour.
 9. The method of claim 1 whereinthe O₂ concentration threshold value is a decrease of between 25% and75% of the O₂ concentration at t_(o).
 10. The method of claim 1 whereincorrelating t_(Threshold) to a viable aerobic microbe count in thesample prior to incubation is accomplished by solving the linearequation for x.
 11. The method of claim 1 wherein correlatingt_(Threshold) to a viable aerobic microbe count in the sample prior toincubation is accomplished by use of a look-up table.
 12. The method ofclaim 1 wherein the x-intercept is an experimental value.
 13. The methodof claim 1 wherein the x-intercept is an estimated value between 6 and10.
 14. The method of claim 1 wherein the sample is incubated in ahermetically sealed chamber in the presence of an oxygen sensitivephotoluminescent dye and O₂ concentration of the incubating sample isascertained by measuring fluorescence of the oxygen sensitive dye. 15.The method of claim 14 wherein luminescent lifetime of the oxygensensitive dye changes in response to changes in the O₂ concentration ofthe incubating sample.
 16. The method of claim 14 wherein theoxygen-sensitive photoluminescent dye is embedded within anoxygen-permeable hydrophobic polymer matrix in physical contact with thesample.
 17. The method of claim 14 wherein the oxygen-sensitivephotoluminescent dye is dissolved in the sample.